In these various fields, the detectors used commonly consist of at least two tanks. One tank, called the reference tank, contains a perfectly identified "control" medium, whereas a (generally liquid) medium certain properties of which vary according to its composition and which is to be analyzed circulates in the other tank.
High performance liquid chromatographs most often utilize these types of detectors, be they photometric, refractometric or spectrophotometric.
Photometers are advantageous in that they afford a great sensitivity and a high stability. They may be used in all the cases in which the eluates to be detected absorb the light within the wavelengths ranging between 190 and 700 nm. However, these photometers have major drawbacks: they are not universal and, in the same analysis, it is possible to meet with eluates which absorb the light at different wavelengths or which even do practically not absorb it, which entails the drawback of letting impurities go unnoticed, notably in preparative chromatography.
Moreover, in the case of preparative chromatography, the photometer is rapidly saturated around an optical density of the order of two.
This drawback may be decreased by dividing for example the length of the optical path in the tank by ten, but this entails a further loss of sensitivity.
The main advantage of refractometers is that they are practically universal. Most of the devices which are currently marketed are light beam deviation through double prismatic circulating vessel refractometers. In these detectors, a light source casts a beam onto a double photodetector after passing successively through a diaphragm, possibly concentrating lenses, a rotating glass strip with parallel faces for adjusting the optical zero of the device, i.e. for balancing the light intensity lighting up the two photodetectors, a double prismatic tank, one for a reference liquid and the other for the phase to be analyzed.
When the refractive index of the latter varies, the prismatic section of the two successive tanks is such that the beam deviates from one photodetector towards the other, according to the sign of the difference of the indexes between the two tanks. However, in some cases, notably in preparative chromatography, where high concentrations may be found in one of the two tanks, or if an elution gradient is created, the index variations may be such that the beam may deviate to the point of saturation of the device, which means that the deviated beam eventually only lights up one of the two cells. The chromatogram is thus clipped and several peaks with a common base may no longer be distinguished.
As it is the case for photometers, these drawbacks may be decreased by reducing the deviations, but here again at the expense of a sensitivity loss. Saturation is then avoided, but the smaller peaks, i.e. impurities, are no longer distinguished in preparative chromatography.
A refractometric system with a monochromatic source has also been proposed, whose beam is divided to pass through two tanks in parallel, for a reference liquid and the phase to be analyzed, then the two beams are reassembled to light up a photodetector. Interferences occur because of the variation of the optical path, on the measurement side, as a function of the index variation. One may consider that the sinusoid followed by the intensity is linear in the neighborhood of the index difference.
Document FR-2,596,526 describes an interferential differential refractometer in which each of the tanks, the reference and the measuring tank, takes independantly part in two independant interferometry systems but is "supplied" with light by the same source (laser for example).
The photometric detection of the two interferometers is performed by two independant photodetectors. It is understandable that, in such a system, each photodetector receives a light intensity which is a sinusoidal function of the difference of the refractive indexes between the reference tank and the measuring tank.
Consequently, if the refractive index varies progressively in very large proportions in one of the tanks, for example in gradient, the corresponding photodetector receives a light intensity of sinusoidal variation, which means that one has a phase information between the measuring and the reference tank which will depend, to within 2k, on the refractive index difference between the two tanks.
If this difference becomes very great, no change occurs, and the instrument, at the detection stage, never reaches saturation point. It is thus possible to refer to infinite measuring dynamics, although the refractive index is a finite quantity.
It has been noticed, for all the differential detectors that have been described, which include notably a measuring tank and a reference tank, that measurings are disturbed by the pressure variations in the tanks.
These pressure variations are due to the fact that the pumps used for circulating the liquid or mobile phase in the chromatography columns are not perfect and some pumps deliver particularly pulsed and unstable flow rates.
Now, it is known that the refractive index of a fluid depends on its density, and therefore on its pressure.
As a consequence of this dependance between pressure and refractive index, these detectors are difficult to use for high sensitivity operations, since flow rate irregularities due to the pumps generate detection signals which become higher than the sought signals.
Thus, in the known differential refractometers, a flow rate variation, and therefore a pressure variation, affects only the measuring tank since, in the reference tank, the medium is not stressed from outside. This variation, which is in no way compensated by the reference tank, causes an undesirable measuring signal.
Differential spectrophotometers are also affected by this problem, although more indirectly.
The working principle of spectrophotometers is in fact based on the absorption of the ultraviolet light by the products to be analyzed. However, the light beams passing through the reference tank and the measuring tank being not perfectly parallel, part of this light gets lost on the walls of the tank and this loss varies with the refractive index variations of the liquid.
Although the disturbance is lower than in the case of differential refractometers, it still remains a hindrance, notably during high sensitivity operations with low-performance pumps.